Identification of Tropomyosin and Its Immunological Properties from Larvae of Cattle Tick, Boophilus annulatus

Background Boophilus annulatus is an obligate blood feeder tick that can cause great losses in animals due to anemia and its ability to injure its host skin directly. The aim of this study was identification of cattle humoral immune response to some tick proteins during experimental infestation. Methods Immune sera against tick were collected from experimentally infested cattle with ticks. One and two-dimensional electrophoresis and Western blotting methods were used for the detection of immunogenic proteins in larval tick extract and eight of these proteins were identified by MALDITOF and MALDI-TOF-TOF mass spectrometry. Results In non-reducing one-dimensional SDS-PAGE, some bounds between 12 to more than 250-kDa appeared. In two-dimensional SDS-PAGE, numerous spot appeared and the identified immunogenic proteins by parallel immunoblotting weighted between 14 and 97 kDa. Amino acid sequences of protein spot with 37-kDa molecular weight had identity to tropomyosin based on Mascot search in NCBI. Conclusion Anti tropomyosin antibodies can be induced in experimentally infested hosts with ticks and it seems that tropomyosin can be useful for the development of anti tick vaccines.


Introduction
oophilus annulatus is an important tick that transmits Babesia bigemina, B. bovis and Anaplasma marginale to cattle. Heavy infestations with the tick cause damages to hides and possibly lead to reduction in the growth rate of cattle (1) B Conventional tick control methods have mainly been based on the use of acaricides; however, the appearance of resistance in tick populations and the presence of chemical residues in nature, meat and milk reveal the need for novel and biological control methods, such as anti tick vaccination (2,3). Although an anti-tick vaccine may be the most promising control method, its development still depends on the identification and characterization of one or more protective tick antigens (4). Tropomyosin is an allergenic, actin-binding protein that regulates the actin organization and is a proposed antigen for vaccine development in several species of the parasite (5). Along with the troponin complex, it associates with actin in muscle fibers and regulates muscle contraction by regulating the binding of myosin. Furthermore, actin filament system (cytoskeleton) is required to perform a remarkable array of functions in eukaryotic cells, including cytokinesis, cell motility, contractile force, intracellular transport, cell morphology and cell size. Tropomyosin isoforms have an important role in generation of actin filament functional diversity (6). A few researches have been carried out on the tick proteomics in Iran, so more knowledge on tick proteins and their characterization can be useful for development of tick vaccine. In the current study, humoral immune responses were investigated in cattle after experimental exposure to B. annulatus and some immunogenic proteins identified using 2Delecterophoresis and Western blotting in unfed larval tick extracts.

Tick collection and rearing
Tick collection and rearing were chosen according to Brown et al. (7)). The adult B. annulatus ticks used in this study were originated from the Band pay of Mazandaran Province (north of Iran) and reared on Hereford calves maintained in the Large Animal Teaching Hospital, Karaj. Eggs were obtained by maintaining fully engorged females at 28°C and 80% relative humidity for oviposition. Hatched larvae were maintained and grown up in the same condition, in Parasitology Laboratory of the Faculty of Veterinary Medicine, University of Tehran. Larvae were stored at -70° C at the 12 th day after hatching until use.

One-dimensional electrophoresis
Initial studies included 12% tris-glycine/SDS gels and running buffers under a non-reducing condition. All separations of tick larvae proteins were performed using a mini gel electrophoresis system (BioRad, USA). Protocols and methods were as described by Lamelli (8). B. annulatus unfed larvae were grounded and homogenized in PBS, then 6 µl (10 µg) of the protein were diluted two folds in SDS-PAGE sample buffer and then electrophoresed on gel at 80 V for 90 min. Coomassi and silver stains were used to visualize proteins.

Immunoblotting
Immunoblotting was carried out according to original protocols by Wang et al. (10) with some modifications. Larval tick protein extracts were separated in 12% SDS-PAGE gels and transferred to nitrocellulose membranes using a wet system (BioRad, USA) in transfer buffer. The membrane blocked 45 min with PBS containing 2.5% Tween 20 and all the washing steps were carried out with PBS containing 0.05% Tween 20 (3 × 5 min). The membrane was incubated one hour in serum derived from cattle experimentally infested with B. annulatus larvae (1/50 in PBS containing 0.05% Tween 20) at room temperature; then, washed and incubated with anti-bovine IgG coupled to horse radish peroxidase (1/1000 in PBS) for 30 min at room temperature. After washing, the color development step was performed with diamino benzidine containing H 2 O 2 .

Mass Spectrometry
Based on the Western blot results, eight of the immunogenic protein spots were excised from parallel 2-D gel stained with Coomassi blue, and subjected to York University in England for MALDI-TOF and MALDI TOF/TOF mass spectrometry. The spectral data were compared with data annotated to NCBI database using Mascot software.

1-D SDS-PAGE
After electrophoresis under non-reducing conditions, eight sharp bands with molecular weights between 40 and more than 250 (kDa) were detected (Fig. 1). In non-reducing SDS-PAGE with silver staining, more bands between 12 and more than 250 kDa were detected (Fig. 2).

2-D SDS-PAGE and immunoblotting
B. annulatus larval extract proteins (300 µg) were separated using isoelectric focusing on a non-linear immobilized pH gradient (3)(4)(5)(6)(7)(8)(9)(10) and second dimension electrophoresis on a 12% gel.  After staining, numerous spots were detected (Fig. 3). B. annulatus larval extract was reacted with collected sera from cattle, which have been experimentally infested with tick six times. Some of the spots related to Coomassi blue stained 2-D gels were detected as immunogenic spots based on the results of 2-D Western blotting by immune sera. Eight immunogenic spots (nos 1-8), between 14 and 97 kDa, were analyzed by MALDI-TOF and MALDI TOF/TOF mass spectrometry (Fig. 4).  A little or no reactive spots were observed in blots incubated with non-infected cattle sera as control (Fig. 5).

Mascot Search Results
The MALDI-TOF and MALDI TOF/TOF analysis against the NCBI database restricted to Metazoa provided evidence that spot number six is homologus of B. micropolus tropomyosin. Four protein spots (1,2,4,7) were related to vitellogenin. Spot number 5 had a single peptide match to hypothetical protein ISCW001652 (Ixodes scapularis). Two protein spots (3,8) were not identifiable, suggesting that they may be novel molecules. Protein view for spot number six is shown in Fig.  6. Peptide sequence search results using MS/MS for protein number 6 are shown in Table 1.  Predicted mass values determined by database search algorithms, estimated from their amino acid sequences. c Protein score is derived from ion scores (sum of ion scores). Ions score is -10 Log (P), where P is the probability that observed match is a random event.

Discussion
Boophilus annulatus is a single-host tick that causes important losses to bovine herds annually. Protective antigens are being investigated in order to develop vaccines to avoid the use of acaricides. Therefore, proteome analysis of B. annulatus larvae can help to candidate targets to develop novel control strategies. Two-dimensional electrophoresis is one of the appropriate methods, which can be used for the separation, and quantification of proteins (11). Although 1-D electrophoresis does not have the same resolving capacity, it can be useful in conjunction with 2-D electrophoresis. In this study, for the first time we identified immunogenic proteins in B.annulatus by a combination of 2-D electrophoresis and Western blotting. One of the eight spots (with 37 kDa molecular weight) that submitted for MALDI-TOF MS/MS was identified as trpomyosin, which reacted with immune cattle serum. As shown in Table 1, tropomyosin proteins from other invertebrates matched with this protein.
There are multiple tropomyosin isoforms in invertebrates, which suggests the complexity of tropomyosin function. Tropomyosin has a wide distribution in muscle and non-muscle tissues. Vertebrate muscle tropomyosin is a dimer molecule arranged in an in-parallel, inregister coiled coil (12). Sometimes, muscletype tropomyosin is expressed in non-muscle tissues. Some investigations have suggested that tropomyosin isoform expression is highly regulated during the development and between different cells (13). The amount and isoform type of tropomyosin in actin filaments are important for specializing the actin function (6). In order to identify functional genes, Zhao et al. (14)) constructed cDNA expression library from mRNA extracted from larval Haemaphysalis qinghaiensis ticks. They found 11 positive clones from the library through immunoscreening of the library using the polyclonal antibody generated in rabbits with larval tick protein extracts. The amino acid sequences of one of these clones shared 94%, 96% and 80% identities with the tropomyosin mRNAs of H. longicornis, B. micropolus and Aleuroglyphus ovatus ticks, respectively (NCBI/ BLASTIN). They suggested that tro-pomyosin might be a vaccine candidate for H. qinghaiensis. Nisbet et al. (5) characterized tropomyosin, its immunogenicity and tissue distribution in poultry red mites. Encoded proteins from cDNA showed 89% and 88% identities to tropomyosins from B. micropolus and Haemaphysalid longicornis respectively, and 85% identity to the house dust mite tropomyosin. They also showed the mouse antibodies against house dust mite tropomyosin reacted with a band of 38 kDa on Western blots of D. gallinae extract, consistent with the molecular masses of acarin tropomyosin. The IgY component of the sera from infested hens in Western blot of D. gallinae extract did not bind to tropomyosin, but reacted with a number of mite proteins. Therefore, they concluded that antitropomyosin antibodies were not induced in hens after the natural exposure to mites. Their immunolocalization study of tropomyosin in mites showed a vast distribution of the molecule in mite tissues. Aki et al. (15) showed that tropomyosin is an allergenic protein and reacts with specific IgE in sera of humans allergic to house dust mite. They have also shown that tropomyosin of the sheep scab mite Psoroptes ovis can induce IgE and IgG responses in sheep during infestation (16). Hartmann et al. (17) showed that immunization of jirds with a purified protein (41 kDa tropomyosin-like protein) from the filarial nematode Acanthocheilonema viteae caused the reduction of adult worm burden by > 60% and the circulating microfilariae by up to 93%. The role of tropomyosin in host resistance to microfilaria in onchocerciasis was investigated by Rosalind et al. (18) and Folkard et al. (19). They concluded that muscle isoform of tropomyosin from dying or dead microfilaria may induce a humoral response that can cross react with a non-muscle form, which can injure the parasite most likely via antibody-dependent cell toxicity. This phenomenon was supported by the attachment of anti-tropomyosin antibodies to the cuticle of microfilariae, as confirmed by immunoelectron microscopy. Therefore, they suggested that anti-tropomyosin antibodies, in parallel with antibody independent mechanisms, could influence microfilarial densities. In current study, tropomyosin of B. annulatus larval extract migrated with an apparent molecular mass of 37 kDa on SDS-PAGE; however, the predicted size of this isoform was 32.9 KDa. According to Untalan et al. this difference in molecular masses could be attributed to post-translational modifications, multiple protein isoforms and different protein compositions (20). These data were similar to data published by Rosalind et al. (18), who showed a tropomyosin isoform from Onchocerca vulvulus with an apparent molecular mass of 42 kDa on SDS-PAGE, but with predicted size of 33.2 KDa. They also suggested that the protein might be post-translationally modified. Moreover, they obtained sequences, which shared 91% identity with tropomyosin from other nematodes.

Conclusion
Antitropomyosin antibodies are induced in cattle after experimental exposure to ticks and may help to protect against tick infestation; therefore, tropomyosin can be considered as a antigen for anti tick vaccine.